Solid-phase clonal amplification and related methods

ABSTRACT

The present invention provides methods and compositions for analyzing nucleic acid sequences. In some aspects, the methods utilize clonal objects, such as DNA balls, that have been captured on beads. Using the methods described here, compositions are fabricated wherein a bead and one clonal object are affinity bound or hybridized to each other through an affinity binding patch or hybridization patch on the surface of the bead. The invention also provides a population of beads having affinity bound or hybridized clonal objects at a ratio of 1:1. The invention additionally provides methods for amplifying a target nucleic acid molecule utilizing the compositions described herein.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods foranalyzing nucleic acid sequences, and more specifically to compositionshaving a solid substrate and a clonal object for analysis includingmethods of fabricating and using the compositions.

BACKGROUND OF THE INVENTION

The task of cataloguing human genetic variation and correlating thisvariation with susceptibility to disease is daunting and expensive. Asingle genome sequence has a price tag of approximately $50,000. Adrastic reduction in this cost is imperative for advancing theunderstanding of health and disease. The near term goal in genomicsanalysis is to resequence the human genome at a cost of approximately$1,000 dollars. A reduction in sequencing costs will require a number oftechnical advances in the field. Fortunately, the same basic principlesof readout parallelization and sample multiplexing that proved sopowerful for gene expression and SNP genotyping analysis are also beingsuccessfully applied to large-scale sequencing. Technical advances thatcould reduce the cost of genome analysis include: (1) librarygeneration; (2) highly-parallel clonal amplification and analysis; (3)development of robust cycle sequencing biochemistry; (4) development ofultrafast imaging technology; and (5) development of algorithms forsequence assembly from short reads.

The ability to specify the content of the DNA library in a targetedmanner is extremely useful for a number of applications. In particular,the ability to resequence all exons in the cancer genome would greatlyfacilitate the discovery of new cancer genes. The comprehensiveresequencing of cancer genomes is a major objective of the Cancer GenomeAtlas Project (cancergenome.nih.gov/index.asp) and would greatly benefitfrom a reduction in sequencing price. Unfortunately, creating a targetedlibrary of the 250,000 exons from the genome is cumbersome using currentmethods. The approach of single-plex PCR for each exon is clearly costprohibitive. As such, parallelization of the sample preparation is ofparamount importance in reducing sequencing costs.

In addition to library generation, the creation of clonal amplificationsin a highly-parallel manner is also important for cost-effectivesequencing. Sequencing is generally performed on clonal populations ofDNA molecules traditionally prepared from plasmids grown from pickingindividual bacterial colonies. In the human genome project, each clonewas individually picked, grown-up, and the DNA extracted or amplifiedout of the clone. In recent years, there have been a number ofinnovations to enable highly-parallelized analysis of DNA clonesparticularly using array-based approaches. In the simplest approach, thelibrary can be analyzed at the single molecule level which by its verynature is clonal. The major advantage of single molecule sequencing isthat cyclic sequencing can occur asynchronously since each molecule isread out individually. In contrast, analysis of clonal amplificationsrequires near quantitative completion of each sequencing cycle,otherwise background noise progressively grows with each ensuing cycleseverely limiting read length. As such, clonal analysis places a biggerburden on the robustness of the sequencing biochemistry and maypotentially limit read lengths.

Thus, there exists a need to develop methods to improve genomicsanalysis and provide more cost effective methods for sequence analysis.The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for analyzingnucleic acid sequences. In some embodiments, the methods utilize clonalobjects, such as DNA balls, that have been captured on beads.Embodiments of the invention provide compositions having a bead and oneclonal object, wherein the clonal object is affinity bound or hybridizedto the bead, for example, through patch on the surface of the bead, suchas an affinity binding patch or a hybridization patch. In some aspects,the patch includes a plurality of polynucleotides attached to a singleregion on the surface of the bead. Embodiments of the invention alsoprovide a population of beads having affinity bound or hybridized clonalobjects. In particular embodiments, each of the clonal objects areaffinity bound or hybridized to the beads through a patch on the surfaceof each bead, such as an affinity binding patch or a hybridizationpatch. The ratio of beads to bound or hybridized clonal objects in thepopulation can be 1:1. In particular embodiments, no more than oneclonal object is bound or hybridized to any given bead in thepopulation.

Using the methods described herein, compositions can be fabricatedwherein a bead and one clonal object are affinity bound or hybridized toeach other through attachment to a patch on the surface of the bead.Embodiments of the invention can provide a method of fabricating anaffinity binding patch on a bead by providing a bead having a pluralityof capture moieties; providing a solid surface having a plurality ofcapture-complement moieties, wherein the capture-complement moietiesfurther comprise a cleavable moiety and an affinity ligand; specificallybinding the capture moieties to the capture-complement moieties, therebyforming an immobilized bead on the solid surface; and cleaving thecleavable moiety so as to retain the affinity ligand on the bead,thereby fabricating an affinity binding patch on the bead. In particularembodiments, the capture moieties or the capture-complement moieties orboth include capture sequences of polynucleotides. Accordingly,embodiments of the invention can provide a method of fabricating anaffinity binding patch on a bead by providing a bead having a pluralityof first polynucleotides attached to the surface of the bead, whereinthe first polynucleotides each have a capture sequence; providing asolid surface having a plurality of second polynucleotides attached tothe solid surface, wherein the second polynucleotides each have acapture-complement sequence, a cleavable moiety and an affinity ligand;hybridizing the capture sequences of the first polynucleotides to thecapture-complement sequences of the second polynucleotides, therebyforming an immobilized bead on the solid surface; and cleaving thesecond polynucleotides at the cleavable moiety so as to retain theaffinity ligand on the second plurality of polynucleotides, therebyfabricating an affinity binding patch on the bead. In some aspects, themethod further includes fabricating one clonal object bound to theaffinity binding patch by contacting the affinity ligand with a bindingagent, wherein the binding agent has two or more binding sites, andbinding one clonal object to the binding agent through a second affinityligand on the clonal object, wherein the one clonal object has a singletandemly repeated target nucleic acid molecule, thereby fabricating oneclonal object bound to the affinity binding patch.

Embodiments of the invention can provide a method of fabricating a beadhaving one clonal object by providing a bead having a plurality of firstcapture moieties; providing a solid surface having a plurality of secondcapture moieties patterned into patches on the surface, wherein thesecond capture moieties each have a cleavable moiety, wherein one clonalobject is bound to one patch on the surface via one or more of thesecond capture moieties, wherein the one clonal object has a singletandemly repeated target nucleic acid molecule; specifically binding thefirst capture moiety to the clonal object, thereby forming animmobilized bead on the solid surface, and cleaving the cleavable moietyso as to retain the clonal object, thereby fabricating a bead having oneclonal object. In particular embodiments, the capture moieties comprisepolynucleotides. Accordingly, embodiments of the invention can provide amethod of fabricating a bead having one clonal object by providing abead having a plurality of first polynucleotides; providing a solidsurface having a plurality of second polynucleotides patterned intopatches on the surface, wherein the second polynucleotides each have acleavable moiety, wherein one clonal object is hybridized to onepolynucleotide patch on the surface, wherein the one clonal object has asingle tandemly repeated target nucleic acid molecule; hybridizing thefirst polynucleotides to the clonal object, thereby forming animmobilized bead on the solid surface, and cleaving the secondpolynucleotides at the cleavable moiety so as to retain the clonalobject, thereby fabricating a bead having one clonal object.

Embodiments of the invention can provide a method of fabricating ahybridization patch on a bead by providing a bead having a plurality offirst polynucleotides attached to the surface of the bead, wherein thefirst polynucleotides each have a first capture sequence, providing asolid surface having a plurality of second polynucleotides attached tothe solid surface, wherein the second polynucleotides each have a firstcapture-complement sequence and a second capture-complement sequence,hybridizing the first capture sequences of the first polynucleotides tothe first capture-complement sequence of the second polynucleotides,thereby forming an immobilized bead on the solid surface, and extendingthe first polynucleotides of the immobilized bead using the secondcapture-complement sequence as a template, thereby fabricating ahybridization patch of extended first polynucleotides on the bead, theextended first polynucleotides having a second capture sequence. In someaspects, the method further includes fabricating one clonal object boundto the patch on the bead by providing a clonal object having the secondcapture-complement sequence, and hybridizing the secondcapture-complement sequence of the clonal object to the second capturesequences of the bead, thereby fabricating one clonal object bound tothe patch on the bead. In some aspects of the method, extending thefirst polynucleotides includes the addition of one or more nucleosidetriphosphates having an affinity ligand, thereby fabricating an affinitybinding patch on the bead.

The invention additionally provides methods of amplifying a targetnucleic acid molecule utilizing the compositions described herein.Embodiments of the invention provide a method of amplifying a targetnucleic acid molecule by placing the compositions or populations ofbeads having affinity bound or hybridized clonal objects describedherein onto a solid surface having microwells, wherein only one bead canspatially fit into one microwell and amplifying the target nucleic acidmolecules in the microwells, thereby forming amplicons. In some aspects,the method further includes sequencing the amplified target nucleic acidmolecules using methods such as sequencing by synthesis, sequencing byligation or sequencing by hybridization, thereby determining the nucleicacid sequence of the target nucleic acid molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures are intended to illustrate broad concepts of the inventionby reference to representative examples for ease of discussion. They arenot intended to limit the scope of the invention by showing one out ofseveral alternate embodiments or by showing or omitting optionalfeatures of the invention.

FIG. 1 shows a schematic depiction of a bead charged with (P1) primerpolynucleotides for hybridization to a plate having complementarybiotinylated (P1′) primer polynucleotides attached to the solid surfaceby a linker having a cleavable moiety. (B) represents the affinityligand biotin. (A) represents the linker having a cleavable moiety.

FIG. 2 shows a schematic depiction of a bead charged with (P1) primerpolynucleotides shown in FIG. 1 after hybridization to the complementarybiotinylated (P1′) primer polynucleotide, crosslinking of the P1 primerto the P1′ primer and cleaving of the linker having a cleavable moietythereby retaining the affinity ligand biotin on the P1′ primer. Alsodepicted is contacting the bead with the binding agent streptavidintetramers. (B) represent the affinity ligand biotin. Square shaded boxesincluding the designation (S) represent the streptavidin tetramers.

FIG. 3 shows schematic depictions of side and face views of a beadhaving an affinity binding patch charged with streptavidin tetramers.

FIG. 4 shows a schematic depiction of single stranded target fragmentligated into a single stranded nucleic acid molecule where the ligationsplint having P1′ and P2 sequences complementary to the single strandedP1 and P2′ sequences serves as the primer for rolling circleamplification (RCA). The primer having a complementary P1′ and P2sequence can optionally be biotinylated (B).

FIG. 5 shows a schematic depiction of a DNA ball having tandemlyrepeated target nucleic acid molecules separated by primer sequences P1′and P2. The primer sequences also include restriction endonuclease (RE)or top nicking endonuclease recognition sequences. The DNA ball canoptionally be biotinylated at either end of the DNA molecule or withinthe DNA ball by incorporation of a biotinylated nucleotide duringamplification.

FIG. 6 shows a schematic depiction of hybridization of oligonucleotideshaving complementary sequences to the site specific endonucleasesequences within the DNA ball thereby providing double strandedstructures necessary for DNA cleavage. Cleavage with double stranded DNAspecific endonucleases will leave the single stranded target sequencesintact.

FIG. 7 shows a schematic depiction of a method for fabricating asepharose microsphere having a single DNA ball.

FIG. 8 shows a schematic depiction of magnetically enriching beadscontacted with magnetic particles.

FIG. 9 shows a schematic depiction of beads contacted with particlesthat are in turn in contact with a patterned surface.

FIG. 10 shows a schematic depiction of mixing bead-bearing droplets withparticle bearing droplets to form droplets containing a bead in contactwith a single particle.

DETAILED DESCRIPTION OF THE INVENTION

In some embodiments, the present invention provides methods andcompositions for analyzing nucleic acid sequences. Embodiments of theinvention provide compositions having a bead and one clonal object,wherein the clonal object is affinity bound or hybridized to the beadthrough a patch on the surface of the beads, wherein the patch includesa plurality of polynucleotides attached to a single region on thesurface of the bead. Embodiments of the invention also provide apopulation of beads having affinity bound or hybridized clonal objects,wherein each of the clonal objects are affinity bound or hybridized tothe beads through a patch on the surface of each of the beads, whereinthe patch includes a plurality of polynucleotides attached to a singleregion on the surface of the beads and wherein the ratio of beads tobound or hybridized clonal objects is 1:1.

In some aspects of the invention, the clonal object has a singletandemly repeated target nucleic acid molecule. Such clonal objectsinclude, for example, DNA balls, particles formed of or with nucleicacids, circular library elements and the like generated using methodsdescribed herein or using any method known to one of skill in the art.Accordingly, embodiments of the invention provide a composition having abead and one clonal object, wherein the one clonal object includes asingle tandemly repeated target nucleic acid molecule and said clonalobject is affinity bound or hybridized to the bead through a patch onthe surface of the bead, wherein the patch includes a plurality ofpolynucleotides attached to a single region on the surface of the bead.Embodiments of the invention also provide a population of beads havingaffinity bound or hybridized clonal objects, wherein each of the clonalobjects includes a single tandemly repeated target nucleic acid moleculeand each of the clonal objects being affinity bound or hybridized to thebeads through a patch on the surface of each bead, wherein the patchincludes a plurality of polynucleotides attached to a single region onthe surface of the beads and wherein the ratio of beads to bound orhybridized clonal objects is 1:1. In some aspects, the invention alsoprovide that the patch on the bead or on each bead of a population canbe a defined area of sufficient size that limits the number of clonalobjects that can bind or hybridize to the bead. For example, in someaspects of the invention, the patch on the surface of the bead is lessthan 1000 nm², or alternatively less than 500 nm², or alternatively lessthan 100 nm².

In accordance with particular embodiments of the invention, beads can befabricated to have a patch on the surface that has reactivity, bindingaffinity or other characteristics that differ from the rest of thesurface of the bead. The patch can be created by selectively modifyingthe area on the surface of the bead that comes into contact with a solidsurface. As set forth in further detail below, the patch can be createdto have one or more affinity ligands so as to create an affinity bindingpatch on the bead. In some embodiments, the affinity ligand can betransferred from a solid surface to a surface of the bead. For example,the affinity ligand can be a ligand or receptor that is attached to anucleic acid that is in turn attached to the solid surface via acleavable linker and transfer can occur via hybridization of the nucleicacid to a complementary sequence on the bead, followed by cleavage fromthe solid phase surface. In some embodiments, the solid surface, or achemical attached thereto, can participate in transfer of an affinityligand from solution to the surface of the bead. For example, theaffinity ligand can be a nucleic acid capture sequence that is encodedby a capture complement sequence present in a solid-surface-attachedpolynucleotide. In this example, a bead-bound polynucleotide can behybridized to the solid-surface-attached polynucleotide and thebead-bound polynucleotide can be extended (for example, by a polymeraseor ligase) using the capture complement sequence as a template such thatthe bead-bound nucleic acid will acquire the capture sequence. In asimilar method, the bead-bound polynucleotide can be extended toincorporate nucleotides or oligonucleotides that have an affinity ligand(such as biotin). In further exemplary embodiments, a solid surface, ora chemical attached thereto, can react with a reactant on the surface ofthe bead to synthesize an affinity ligand on the bead, or the solidsurface, or a chemical attached thereto, can expose or deprotect anaffinity ligand on the surface of the bead.

Beads having an affinity binding patch can be useful for creating aclonal library of target nucleic acids. The affinity binding patchallows efficient creation of a collection of beads in which each beadcarries a single target nucleic acid sequence (whether the singlesequence is present in a single copy or multiple copies) and in whichfew or no beads are devoid of a target nucleic acid sequence. Asexemplified in several embodiments below, a population of target nucleicacids can be contacted with beads under conditions wherein only a singletarget nucleic acid is capable of binding to the affinity patch suchthat no more than one target nucleic acid is attached to any given bead.For example, the target nucleic acids can be in the form of clonalobjects, such as DNA balls or nucleic acids in particle form, thatindividually bind to a patch so as to sterically exclude subsequentclonal objects from binding to the same patch. Since only a singletarget nucleic acid is capable of binding to any bead, saturatingamounts of target nucleic acid can be contacted with the beads to drivethe reaction toward yielding a population of beads in which most or allof the individual beads have an attached target nucleic acid. Thisability to drive the reaction to a state in which few to no beads lack atarget nucleic acid (i.e. “blank” beads) provides advantages over othermethods that rely on limiting amounts of nucleic acid to yield a Poissondistribution of beads bearing a single target nucleic acid because thelatter methods, although yielding few beads with more than one targetnucleic acid, end up having a large number of blank beads. In manyembodiments blank beads are undesirable since they can consume time andresources in downstream analyses. A clonal library created using themethods set forth herein can provide separation of individual sequencesand efficient use of beads to provide benefits for multiplex analyses ofcomplex nucleic acid samples such as genomes. Several exemplaryapplications including nucleic acid analysis as set forth in furtherdetail below.

The terms “microsphere” and “bead” are used interchangeably and mean asmall body made of a rigid or semi-rigid material. The body can have ashape characterized, for example, as a sphere, oval, microsphere, orother recognized particle shape whether having regular or irregulardimensions. In particular embodiments the small body has a curvedsurface. Populations of microspheres or other small bodies can be usedfor attachment of populations of capture probes, amplicons, DNA balls orother nucleic acids. The composition of a microsphere can vary,depending for example, on the format, chemistry and/or method ofattachment and/or on the method of nucleic acid synthesis. Exemplarymicrosphere compositions include solid supports, and chemicalfunctionalities imparted thereto, used in polypeptide, polynucleotideand/or organic moiety synthesis. Such compositions include, for example,plastics, ceramics, glass, polystyrene, melamine, methylstyrene, acrylicpolymers, paramagnetic materials, thoria sol, carbon graphite, titaniumdioxide, latex or cross-linked dextrans such as Sepharose™, cellulose,nylon, cross-linked micelles and Teflon™, as well as any other materialswhich can be found described in, for example, “Microsphere DetectionGuide” from Bangs Laboratories, Fishers Ind., which is incorporatedherein by reference. The sizes of the microsphere or bead that can beuseful for the methods described herein can be determined by one ofskill in the art and include, without limitation, about 1 μm, about 2μm, about 3 μm, about 5 μm, about 10 μm, about 20 μm, about 30 μm, about40 μm, about 60 μm, about 100 μm, about 150 μm or about 200 μm indiameter. Other particles can be used in ways similar to those describedherein for beads and microspheres.

A “patch” refers to a small piece, portion or section of a surface towhich one or a plurality of things are attached or hybridized. A patchcan also be referred to by the type of thing that is attached orhybridized to the surface. For example, an affinity binding patch refersto a patch that includes an affinity binding ligand to which acorresponding binding agent can bind or pair. As another example, ahybridization patch refers to a patch that includes a polynucleotide ornucleic acid molecule that can hybridize to another polynucleotide ornucleic acid molecule.

In some aspects of the invention, a patch on the surface of amicrosphere or bead can contain a plurality of polynucleotides attachedto a single region the surface of the microsphere or bead. In someaspects of the invention, a patch can also contain an affinity ligand orbinding agent. In some aspects of the invention, the area of a patch isless than the area of the surface of the microsphere or bead. Theconfiguration of the patch can have any of a variety of shapes orcontours. For example, the surface of the patch can be generally planaror curved. In some aspects, the patch is of sufficient size as to allowbinding of only one clonal object per region. Such a size determinationwill depend on the size of the microsphere or bead, the length ofpolynucleotides attached thereto and the size and/or composition of theclonal object. The shape of the patch is also not limited and willdepend on the shape of the microsphere or bead itself and the methodused to generate the region containing the polynucleotides. The patchcan be a contiguous area on a surface that excludes another area on thesurface. The excluded area can partially surround the patch or,alternatively, the excluded area can entirely surround the patch.Non-limiting examples of the size of the patch described herein includea contiguous area of less than 1000 nm², or alternatively less than 900nm², or alternatively less than 800 nm², or alternatively less than 700nm², or alternatively less than 600 nm², or alternatively less than 500nm², or alternatively less than 400 nm², or alternatively less than 300nm², or alternatively less than 200 nm², or alternatively less than 100nm², or alternatively less than 50 nm². Additionally, the size of thepatch described herein can be a percentage of the overall surface areaof the bead, for example, the size of the patch can be a contiguous areathat is no more than 0.001%, or alternatively no more than 0.005%, oralternatively no more than 0.01%, or alternatively no more than 0.05%,or alternatively no more than 0.1%, or alternatively no more than 0.5%,or alternatively no more than 1.0%, or alternatively no more than 2%, oralternatively no more than 5%, or alternatively no more than 10% oralternative no more than 20%, or alternatively no more than 30%, oralternatively no more than 40%, or alternatively no more than 50% of thesurface area of the bead.

In particular embodiments, a patch can include a surface to which aplurality of things are attached or hybridized. Such things can include,but are not limited to, nucleic acids, polynucleotides,oligonucleotides, probes, target molecules, proteins, ligands,receptors, capture moieties, capture-complement moieties or any of avariety of other molecules set forth herein or otherwise known in theart. It will be understood that the surface within a patch need notnecessarily be entirely occupied by those things, and those things canbe present at any of a variety of quantities, surface densities or localconcentrations within the patch. Typically, an area on a surface that isexcluded from a patch will be devoid of one or more types of things thatare attached to the surface within the patch.

In some aspects of the invention, a plurality of polynucleotides on abead or on each bead of a population include a universal primersequence, a nucleic acid sequence fully or partially complementary tothe polynucleotides attached to a solid surface or a target nucleic acidmolecule. In some aspects, the plurality of polynucleotides on the beador on each bead of a population have a sufficient length which allowsfor immobilization on a solid surface having complementarypolynucleotides or hybridization to a clonal object. Exemplary lengthsinclude, without limitation, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 300, 400 or 500 or more nucleotides. In some aspects, thepolynucleotide length is at least 10 nucleotides, but no more than 1000,900, 800, 700, 600, 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, 50,40, 30, or 20 nucleotides. The invention also provides that lengths inbetween these exemplified sizes can also be sued in the compositions andmethods of the invention.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), genomic DNA, genomic DNAfragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomalRNA, ribozyme, cDNA, recombinant polynucleotide, branchedpolynucleotide, plasmid, vector, isolated DNA of any sequence, isolatedRNA of any sequence, nucleic acid probe, primer or amplified copy of anyof the foregoing. A polynucleotide can comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs. If present,modifications to the nucleotide structure can be imparted before orafter assembly of the polynucleotide. The sequence of nucleotides can beinterrupted by non-nucleotide components. A polynucleotide can befurther modified after polymerization, such as by conjugation with alabeling component. The term also refers to both double- andsingle-stranded molecules. Unless otherwise specified or required, anyembodiment of this invention that makes or uses a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform. Unless otherwise specified or required, a “copy” of apolynucleotide can include the exact copy of the polynucleotide and thecomplementary copy of the polynucleotide in single or double strandedform. In some aspects of the invention, the lengths of the plurality ofpolynucleotides on a bead or solid support are at least 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or 500 or more nucleotides.Alternatively or additionally, the lengths are no more than 1000, 900,800, 700, 600, 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30or 20 nucleotides.

In some aspects of the compositions or methods described herein, thepolynucleotides on a bead or solid support have a “capture sequence”. A“capture sequence” refers to a stretch of nucleotides which whenhybridized to a complementary nucleotide sequence present on apolynucleotide or clonal object gains control of or becomes associatedwith any attached molecule, such as a bead or solid surface. The capturesequence can be continuous or non-continuous and will depend on the anumber of variables including, but not limited to, the size of theattached molecule, the location of the capture sequence within thepolynucleotide and the hybridization methods used. A sequence havingsufficient complementarity to a capture sequence to allow specifichybridization is referred to herein as a “capture-complement sequence.”In particular embodiments, the capture-complement sequence includes asequence that is perfectly complementary to the capture sequence. Thelength of the capture sequence and/or the capture-complement sequencecan be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300,400 or 500 or more nucleotides. Alternatively or additionally, thelengths are no more than 1000, 900, 800, 700, 600, 500, 400, 300, 200,150, 100, 90, 80, 70, 60, 50, 40, 30 or 20 nucleotides. Capturesequences and capture-complement sequences are examples of capturemoieties and capture-complement moieties, respectively. Capturesequences and capture complement sequences can also function as affinityligands. Although several embodiments of the invention are exemplifiedherein with respect to capture sequences and capture-complementsequences, it will be understood that other moieties can be used such asaffinity ligands set forth elsewhere herein or other moieties known inthe art that are capable of specific binding interactions.

A polynucleotide can be composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T). Uracil (U) can also be present, for example, as a naturalreplacement for thymine when the polynucleotide is RNA. Uracil can alsobe used in DNA. Thus, the term “polynucleotide sequence” is thealphabetical representation of a polynucleotide molecule. Thisalphabetical representation can be input into databases in a computerhaving a central processing unit and used for bioinformaticsapplications such as functional genomics, sequence alignment, sequencebuilding and homology searching.

A nucleic acid used in the invention can also include native ornon-native bases. In this regard a native deoxyribonucleic acid can haveone or more bases selected from the group consisting of adenine,thymine, cytosine or guanine and a ribonucleic acid can have one or morebases selected from the group consisting of uracil, adenine, cytosine orguanine. It will be understood that a deoxyribonucleic acid used in themethods or compositions set forth herein can include uracil bases and aribonucleic acid can include a thymine base. Exemplary non-native basesthat can be included in a nucleic acid, whether having a native backboneor analog structure, include, without limitation, inosine, xathanine,hypoxathanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine,5-hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methylguanine, 2-propyl guanine, 2-propyl adenine, 2-thioLiracil,2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine,5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine,6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine,8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituteduracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine,8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine,3-deazaadenine or the like. A particular embodiment can utilizeisocytosine and isoguanine in a nucleic acid in order to reducenon-specific hybridization, as generally described in U.S. Pat. No.5,681,702.

A non-native base used in a nucleic acid of the invention can haveuniversal base pairing activity, wherein it is capable of base pairingwith any other naturally occurring base. Exemplary bases havinguniversal base pairing activity include 3-nitropyrrole and5-nitroindole. Other bases that can be used include those that have basepairing activity with a subset of the naturally occurring bases such asinosine, which basepairs with cytosine, adenine or uracil. Non-nativebases can be modified to include a peptide-linked label. The peptide canbe attached to the base using methods exemplified herein with regard tonative bases. Those skilled in the art will know or be able to determineappropriate methods for attaching peptides based on the reactivities ofthese bases. Alternatively or additionally, oligonucleotides,nucleotides or nucleosides including the above-described non-nativebases can further include reversible blocking groups on the 2′, 3′ or 4′hydroxyl of the sugar moiety.

As used herein, a “universal sequence” refers to a sequence that can beattached, for example, by ligation or other methods disclosed herein, toa nucleic acid sequence, particularly in a population of nucleic acidmolecules, such that the same sequence is attached to a plurality ofdifferent nucleic acid molecules. As used herein, a “plurality” refersto two or more. Such a universal sequence is therefore “common” to themany different nucleic acid molecules to which it is attached. Such auniversal sequence is particularly useful for analyzing multiple samplessimultaneously, as disclosed herein. Examples of universal sequences areuniversal primers and universal priming sites. A universal priming sitecontains a “common priming site” to which an appropriate primer can bindto and which can be utilized as a priming site for synthesis of nucleicacid sequences complementary to the nucleic acid sequence attached tothe universal primer.

A primer sequence can be described as “universal” when the same primersequence appears among a plurality or even all of the polypeptides, sothat a small set of primers can be used for amplification of many or allof the target nucleic acid molecules in the same reaction. The universalpriming sequence can be, for example, between 15 and 30 nucleotides inlength in some embodiments, and between 17 and 20 nucleotides in otherembodiments.

In some aspects of the invention, a plurality of polynucleotides on abead or on each bead of a population, include one or more affinityligands attached to the polynucleotides. Non-limiting examples ofaffinity ligands useful in the invention are biotin, imino-biotin, anantibody or functional fragment thereof, an aptamer, a Spiegelmer, areceptor, avidin, streptavidin, neutravidin, a nucleic acid, a peptideor a peptide nucleic acid. Methods of attaching an affinity ligand tothe plurality of polynucleotides will depend on the affinity ligandbeing used, the location of the affinity ligand on the polynucleotide,i.e. 5′ and 3′ ends or within the polynucleotides, and the point ofattachment on the polynucleotide, i.e. the sugar backbone, phosphategroup or base. Methods for attaching an affinity ligand to apolynucleotide are well know to one skilled in the art. One exampleincludes incorporation of biotinylated nucleotides by terminaldeoxynucleotidyl transferase or using DNA polymerase (Flickinger et al.,Nucleic Acids Research Vol. 20(9):2382 (1992) and Tabor and Boyle, Curr.Protoc. Immunol. Chapter 10:Unit 10.10). Biotinylated dNTPs arecommercially available from a number of sources including (LifeTechnologies—Carlsbad, Calif.) and (Roche DiagnositcsCorporation—Indianapolis, Ind.). Another example of attaching anaffinity ligand to a polynucleotide includes induction of cross-linkagesof polynucleotide-proteins by ultraviolet irradiation (Budowsky et al.,European Journal of Biochemistry 159(1):95-101 (1986)). It is alsopossible to use a primer having one or more affinity ligands such thatthe products of an extension or amplification reaction using the primerwill include the one or more affinity ligands.

In some aspects of the invention, the affinity ligands attached to thepolynucleotides are capable of binding to one or more binding agent.Non-limiting examples of binding agents useful in the invention includeavidin, streptavidin, neutravidin, biotin, imino-biotin, an antibody orfunctional fragment thereof, an aptamer, a Spiegelmer, a receptor, anucleic acid, a peptide and a peptide nucleic acid. A particularlyuseful binding agent is streptavidin, a tetrameric protein with a biotinbinding-site in each monomeric unit, which makes it capable of bindingto one, two, three or four biotin molecules. Other binding agents havingtwo or more binding sites for a ligand of interest are also particularlyuseful.

The term “affinity” refers to how tightly a ligand, such as a chemicalcompound, binds to a binding agent, such as a protein. The affinitybetween a ligand and a binding agent can often be expressed by thedissociation constant between the ligand and the binding agent. Theligand-binding agent affinities can be influenced by a number ofdifferent interactions such as non-covalent intermolecular interactionsincluding, but not limited to, hydrogen bonding, electrostaticinteractions, hydrophobicity and Van der Waals forces. Exemplaryligand-binding agent pairs that can be used in the invention include,but are not limited to, immunoglobulins (i.e. antibodies) or functionalfragments thereof (i.e. Fab, F(ab)₂ Fv, and single chain Fv (scFv)) andantigen (i.e. protein or peptide); biotin and avidin or analoguesthereof having specificity for avidin, such as imino-biotin, or havingspecificity for biotin, such as streptavidin or neutravidin; peptide andpeptide interactions, such as a ligand and a receptor; peptide nucleicacids (PNA) and nucleic acids, such as DNA or RNA; and carbohydrates andlectins. The binding pairs set forth above can also be used to attachclonal objects such as DNA balls to beads.

The term “hybridized” or “hybridization” refers to a reaction in whichone or more polynucleotides react to form a complex that is stabilizedvia hydrogen bonding between the bases of the nucleotide residues. Thehydrogen bonding may occur by Watson-Crick base pairing, Hoogsteinbinding, or in any other sequence-specific manner. The complex maycomprise two strands forming a duplex structure, three or more strandsforming a multi-stranded complex, a single self-hybridizing strand, orany combination of these. A hybridization reaction may constitute a stepin a more extensive process, such as the initiation of a PCR reaction,or the enzymatic cleavage of a polynucleotide by a ribozyme.

Hybridization reactions can be performed under conditions of different“stringency”. In general, a low stringency hybridization reaction iscarried out at about 40° C. in 10×SSC or a solution of equivalent ionicstrength/temperature. A moderate stringency hybridization is typicallyperformed at about 50° C. in 6×SSC, and a high stringency hybridizationreaction is generally performed at about 60° C. in 1×SSC. Hybridizationreactions can also be performed under “physiological conditions” whichis well known to one of skill in the art. A non-limiting example of aphysiological condition is the temperature, ionic strength, pH andconcentration of Mg+ normally found in a cell.

When hybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides, the reaction is called “annealing” andthose polynucleotides are described as “complementary”. Adouble-stranded polynucleotide can be complementary or homologous toanother polynucleotide, if hybridization can occur between one of thestrands of the first polynucleotide and the second. Complementarity orhomology (the degree that one polynucleotide is complementary withanother) is quantifiable in terms of the proportion of bases in opposingstrands that are expected to form hydrogen bonding with each other,according to generally accepted base-pairing rules.

As used herein a “binding agent” refers to a molecule that is capable ofbinding to one or more affinity ligands as described herein. A bindingagent can be attached to a polynucleotide to allow detection orisolation of the nucleic acid via specific affinity to an affinityligand. A binding agent can be a sequence or sequence region of thepolynucleotide. Specific affinity between two binding partners isunderstood to mean preferential binding of one partner to anothercompared to binding of the partner to other components or contaminantsin the system. Binding partners that are specifically bound typicallyremain bound under the detection or separation conditions describedherein, including wash steps to remove non-specific binding. Dependingupon the particular binding conditions used, the dissociation constantsof the pair can be, for example, less than about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹,10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³, or 10⁻¹⁴ M.

A further example of a nucleic acid with an analog structure that isuseful in the invention is a peptide nucleic acid (PNA). The backbone ofa PNA is substantially non-ionic under neutral conditions, in contrastto the highly charged phosphodiester backbone of naturally occurringnucleic acids. This provides two non-limiting advantages. First, the PNAbackbone exhibits improved hybridization kinetics. Secondly, PNAs havelarger changes in the melting temperature (Tm) for mismatched versusperfectly matched basepairs. DNA and RNA typically exhibit a 2-4° C.drop in Tm for an internal mismatch. With the non-ionic PNA backbone,the drop is closer to 7-9° C. This can provide for better sequencediscrimination. Similarly, due to their non-ionic nature, hybridizationof the bases attached to these backbones is relatively insensitive tosalt concentration. A PNA or monomer unit used to synthesize PNA caninclude a base having a peptide linked label. In such cases, an enzymeused to cleave the peptide linker will generally be unreactive towardthe PNA backbone.

In some aspects of the invention, a clonal object that is affinity boundor hybridized to a bead or each bead of a population is composed of asingle tandemly repeated target nucleic acid molecule, such as, but notlimited to, a DNA ball, a circular library element or the like. Methodsfor generating such clonal objects are well known to one skilled in theart and include the methods described herein such as rolling circleamplification and DNA ligation. The clonal objects of the invention caninclude multiple copies of the tandemly repeated target nucleic acidmolecule. Multiple copies of the target nucleic acid can be useful forcertain aspects of sequence analysis, such as providing sufficientstarting material for clonal amplification, which in turn allows for aclear signal above any detectable background during sequencing. In someaspects of the invention, the clonal object includes at least 100, oralternatively at least 200, or alternatively at least 500, oralternatively at least 1000, or alternatively at least 2000, oralternatively at least 3000, or alternatively at least 4000, oralternatively at least 5000, or alternatively at least 6000, oralternatively at least 7000, or alternatively at least 8000, oralternatively at least 9000 or alternatively at least 10,000 copies ofthe target nucleic acid molecule.

It is understood that the size of the clonal object can be affected bythe length of the target nucleic acid molecule, the number of copies ofthe target nucleic acid molecule, the presence or absence of interveningprimer, probe or endonuclease recognition sequences and theenvironmental conditions, which can affect the compaction of the clonalobject. Accordingly, in some aspects of the invention, the size of theclonal object is relative to the size of the affinity patch on thesurface of the bead or each bead of a population. In other words, inorder to attach a single clonal object to a bead, the size of the clonalobject is such that once bound to a patch of polynucleotides on thebead, another clonal object is excluded from binding to the bead. Forexample, the diameter or width of the clonal object can be larger thanthe diameter or width of the patch on surface of the bead. For example,a clonal object can have a diameter that is larger than 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100% or more of the diameter of the patchon the surface of the bead. In some embodiments the diameter of theclonal object can be within a range that is smaller than the diameter ofthe patch but large enough to exclude binding of a second clonal objectof similar size. Exemplary ranges include at least about 50%, 100%,200%, 400% or 500% of the diameter of the patch.

In some aspects of the invention, the size of the bead is relative tothe size of the clonal object, such as, the diameter or width of thebead is equal to the diameter or with of the clonal object.Alternatively, the diameter or width of the bead is larger than thediameter or width of the clonal object, such as, at least 10%, oralternatively at least 20%, or alternatively at least 50%, oralternatively at least 75%, or alternatively at least 100%, oralternatively at least 2 time or alternatively at least 5 times, oralternatively at least 10 times, or alternatively at least 100 times oralternatively at least 500 times, or alternatively at least 1000 times,or alternatively at least 5,000 times larger. In some aspects of theinvention, the clonal object has a diameter of 0.1 μm, or alternatively0.2 μm, or alternatively 0.5 μm, or alternatively 1 μm, or alternatively2 μm, or alternatively 3 μm, or alternatively 4 μm, or alternatively 5μm.

As used herein, the term “clonal object” refers to a particle having anucleic acid sequence in one or more copies. An exemplary clonal objectis a nucleic acid that as been amplified from a target nucleic acidmolecule and in some aspects has a single tandemly repeated sequence ofthe target nucleic acid molecule. Such tandemly repeated sequences mayalso be separated with non-target nucleic acids, such as primer bindingsites, endonuclease recognition sites, nucleotides linked to affinityligands or the like. In particular embodiments a clonal object can be aDNA ball, for example, formed by rolling circle amplification. Methodsof generating a clonal object are well known to one of skill in the artand exemplary methods are also described herein. As used herein, a“clonal object” can be synthesized using an amplification technique andthus is also referred to herein as an amplicon. Accordingly, an ampliconis the nucleic acid product of an amplification reaction.

A method for generating an array of amplified nucleic acid sequences caninclude the step of attaching at least one second universal primerhaving a second common priming site to a plurality of sample nucleicacid molecules, thereby attaching a first universal primer and a seconduniversal primer to a sample nucleic acid molecule of the plurality ofsample nucleic acid molecules. In a particular embodiment, the firstuniversal primer and the second universal primer can be attached torespective ends of each nucleic acid in the plurality of sample nucleicacid molecules by ligation.

In embodiments that include ligation of a first double stranded nucleicacid end to a second double stranded nucleic acid end, the ends to beligated can be blunt or can have complementary single strandedoverhangs. The use of complementary overhangs generally provides anadded measure of specificity over blunt end methods because conditionscan be used in which non-complementary sequences will not ligate.Further specificity can be attained by partially filling in one overhangend to make it complementary to another end. This fill in method can beused to disfavor unwanted ligation between nucleic acids in a samplethat were generated with the same restriction enzyme.

An amplicon typically contains multiple, tandem copies of thecircularized nucleic acid molecule of the corresponding sample nucleicacid. That is, each amplicon contains multiple, tandem copies of asingle sample nucleic acid molecule, which was circularized. The numberof copies can be varied by appropriate modification of the amplificationreaction including, for example, varying the number of amplificationcycles run, using polymerases of varying processivity in theamplification reaction and/or varying the length of time that theamplification reaction is run, as well as modification of otherconditions known in the art to influence amplification yield. Generally,the number of copies of a nucleic acid in an amplicon is at least 100,200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 and10,000 copies, and can be varied depending on the particularapplication. As disclosed herein, one particular form of an amplicon isas a nucleic acid “ball” having desired dimensions. The number of copiesof the nucleic acid molecule can therefore provide a desired size of anucleic acid “ball” or a sufficient number of copies for efficientsubsequent analysis of the amplicon, for example, sequencing.

The terms “target nucleic acid molecule,” “target nucleic acid sequence”or any grammatical equivalent thereof, refers to nucleic acid moleculesor sequences that are desired to be detected, sequenced or otherwiseanalyzed. Any of a variety of desired target nucleic acid sequences canbe utilized, including but not limited to exons, or nucleic acidsequences complementary thereto; cDNA sequences, or nucleic acidsequences complementary thereto; untranslated regions (UTRs) or nucleicacids complementary thereto; promoter and/or enhancer regions, ornucleic acid sequences complementary thereto; evolutionary conservedregions (ECRs), or nucleic acid sequences complementary thereto;transcribed genomic regions, or nucleic acid sequences complementarythereto. Any of a variety of methods can be used to obtain targetednucleic acid sequences, as disclosed herein. Such methods include, butare not limited to, obtaining a targeted nucleic acid molecule usinghybridization-extension capture enrichment; using targeted restrictionsites, for example, using an oligonucleotide engineered with a hairpinhaving a Type IIS restriction enzyme site such as a FokI restrictionenzyme site and a locus-specific region; using locus-specifichyperbranched rolling circle amplification; using random-locus-specificprimer amplification; using multiplex emulsion PCR; using multiplexbridge PCR; using padlock probe amplification; and using mini-librariesfrom targeted libraries, as disclosed herein.

As used herein, sample nucleic acid sequences refer to nucleic acidsequences obtained from samples that are desired to be analyzed. Anucleic acid sample that is amplified, sequenced or otherwisemanipulated in a method disclosed herein can be, for example, DNA orRNA. Exemplary DNA species include, but are not limited to, genomic DNA(gDNA), mitochondrial DNA, chloroplast DNA, episomal DNA, viral DNA andcopy DNA (cDNA). One non-limiting example of a subset of genomic DNA isone particular chromosome or one region of a particular chromosome.Exemplary RNA species include, without limitation, coding RNA such asmessenger RNA (mRNA), and non-coding RNA (ncRNA) such as transfer RNA(tRNA), microRNA (miRNA), small nuclear RNA (snRNA) and ribosomal RNA(rRNA). Further species of DNA or RNA include fragments or portions ofthe species listed above or amplified products derived from thesespecies, fragments thereof or portions thereof. The methods describedherein are applicable to the above species encompassing all or part ofthe complement present in a cell. For example, using methods describedherein the sequence of a substantially complete genome can be determinedor the sequence of a substantially complete targeted nucleic acidsequences such as mRNA or cDNA complement of a cell can be determined.

Useful methods for clonal amplification from single molecules includerolling circle amplification (RCA) (Lizardi et al., Nat. Genet.19:225-232 (1998), which is incorporated herein by reference), bridgePCR (Adams and Kron, Method for Performing Amplification of Nucleic Acidwith Two Primers Bound to a Single Solid Support, Mosaic Technologies,Inc. (Winter Hill, Mass.); Whitehead Institute for Biomedical Research,Cambridge, Mass., (1997); Adessi et al., Nucl. Acids Res. 28:E87 (2000);Pemov et al., Nucl. Acids Res. 33:e 11 (2005); or U.S. Pat. No.5,641,658, each of which is incorporated herein by reference), polonygeneration (Mitra et al., Proc. Natl. Acad. Sci. USA 100:5926-5931(2003); Mitra et al., Anal. Biochem. 320:55-65 (2003), each of which isincorporated herein by reference), and clonal amplification on beadsusing emulsions (Dressman et al., Proc. Natl. Acad. Sci. USA100:8817-8822 (2003), which is incorporated herein by reference) orligation to bead-based adapter libraries (Brenner et al., Nat.Biotechnol. 18:630-634 (2000); Brenner et al., Proc. Natl. Acad. Sci.USA 97:1665-1670 (2000)); Reinartz, et al., Brief Funct. GenomicProteomic 1:95-104 (2002), each of which is incorporated herein byreference). The enhanced signal-to-noise ratio provided by clonalamplification more than outweighs the disadvantages of the cyclicsequencing requirement.

In a particularly useful embodiment, amplicons are generated by rollingcircle amplification (RCA), which can be used to generate ampliconshaving multiple copies of a nucleic acid sequence and which can be usedto create nucleic acid “balls,” as disclosed herein. It will beunderstood that these “balls” need not be perfectly spherical and caninclude other globular or packed conformations. In a particularembodiment, RCA is primed using the at least one universal primerattached to the sample nucleic acid molecule.

As disclosed herein, the amplicons can be compacted prior to hybridizingor binding to a bead described herein. Methods of compacting ampliconsare known in the art (for example, as described by Bloomfield, Curr.Opin. Struct. Biol. 6(3): 334-41 (1996), and Drmanac et al., US2007/0099208 A1, each of which is incorporated herein by reference) anddisclosed herein. For example, an alcohol or polyamine such as spermineor spermidine can be used. A compacted nucleic acid will have astructure that is more densely packed than the structure of the nucleicacid in the absence of a compacting agent or compacting condition andthe structure will typically resemble a ball or globule. The generationof such compacted nucleic acid balls is useful for fabricating oneclonal object bound to one bead, as discussed herein in more detail.Various methods can be used to generate balls of a desired size, forexample, using various compacting techniques and/or varying the numberof copies in an amplicon. Generally, the compacted amplicons have anaverage diameter or width ranging from about 0.1 μm to about 5 μm, forexample, about 0.1 μm, about 0.2 μm, about 0.5 μm, about 1 μm, 2 μm,about 3 μm, about 4 μm and about 5 μm.

Embodiments of the invention provide a method of fabricating an affinitybinding patch on a bead by providing a bead having a plurality of firstpolynucleotides attached to the surface of the bead, wherein the firstpolynucleotides each have a capture sequence, providing a solid surfacehaving a plurality of second polynucleotides attached to the solidsurface, wherein the second polynucleotides each have acapture-complement sequence, a cleavable moiety and an affinity ligand,hybridizing the capture sequences of the first polynucleotides to thecapture-complement sequences of the second polynucleotides, therebyforming an immobilized bead on the solid surface, and cleaving thesecond polynucleotides at the cleavable moiety so as to retain theaffinity ligand on the second plurality of polynucleotides, therebyfabricating an affinity binding patch on the bead. In some aspects, themethod further includes fabricating one clonal object bound to theaffinity binding patch by contacting the affinity ligand with a bindingagent, wherein the binding agent has two or more binding sites, andbinding one clonal object to the binding agent through a second affinityligand on the clonal object, wherein the one clonal object has a singletandemly repeated target nucleic acid molecule, thereby fabricating oneclonal object bound to the affinity binding patch.

Embodiments of the invention provide a method of fabricating a beadhaving one clonal object by providing a bead having a plurality of firstpolynucleotides, providing a solid surface having a plurality of secondpolynucleotides patterned into patches on the surface, wherein thesecond polynucleotides each have a cleavable moiety, wherein one clonalobject is hybridized to one polynucleotide patch on the surface, andwherein the one clonal object has a single tandemly repeated targetnucleic acid molecule, hybridizing the first polynucleotides to theclonal object, thereby forming an immobilized bead on the solid surface,and cleaving the second polynucleotides at the cleavable moiety so as toretain the clonal object, thereby fabricating a bead having one clonalobject.

Embodiments of the invention provide a method of fabricating ahybridization patch on a bead by providing a bead having a plurality offirst polynucleotides attached to the surface of the bead, wherein thefirst polynucleotides each have a first capture sequence, providing asolid surface having a plurality of second polynucleotides attached tothe solid surface, wherein the second polynucleotides each have a firstcapture-complement sequence and a second capture-complement sequence,hybridizing the first capture sequences of the first polynucleotides tothe first capture-complement sequence of the second polynucleotides,thereby forming an immobilized bead on the solid surface, and extendingthe first polynucleotides of the immobilized bead using the secondcapture-complement sequence as a template, thereby fabricating ahybridization patch of extended first polynucleotides on the bead,wherein the extended first polynucleotides have a second capturesequence. The second capture sequence can function as an affinity ligandfor attachment of a clonal object. Accordingly, in some aspects, themethod further includes fabricating one clonal object bound to the patchon the bead by providing a clonal object having the secondcapture-complement sequence, or a portion thereof, and hybridizing thesecond capture-complement sequence, or a portion thereof, of the clonalobject to the second capture sequences of the bead, or a portionthereof, thereby fabricating one clonal object bound to the patch on thebead. In some aspects of the method, extending the first polynucleotidesincludes the addition of one or more nucleoside triphosphate having anaffinity ligand, thereby fabricating an affinity binding patch on thebead.

In some aspects, the methods include fabricating one clonal object boundto the affinity binding patch by contacting the affinity ligand with abinding agent, wherein the binding agent has two or more binding sites,and binding one clonal object to the binding agent through a secondaffinity ligand on the clonal object, wherein the one clonal object hasa single tandemly repeated target nucleic acid molecule, therebyfabricating one clonal object bound to the affinity binding patch.

In some aspects, the methods include a step of separating beads that areattached to a clonal object from beads that do not have a clonal object.For example, separation can be carried out based on differences in mass,charge, magnetism or other property imparted by the presence of a clonalobject. By way of further example, separation can be carried out basedon affinity of a clonal object for a receptor such as a nucleic acidbinding protein or an antibody that binds to a ligand on the clonalobject. In another example, beads that are bound to a magnetic clonalobject can be separated from blank beads using magnetic capture.Separation can be carried out prior to a step of fabricating multiplecopies of clonal objects on a solid surface or prior to a step ofcarrying out a nucleic acid analysis using the clonal objects. In someembodiments, a population of beads having attached clonal objects neednot be subjected to a treatment intended to separate out beads lackingclonal objects. In such cases beads can be used directly in a subsequentstep of fabricating multiple copies of clonal objects on a solid surfaceor a step of carrying out a nucleic acid analysis using the clonalobjects.

In some aspects of the above embodiments, the methods further includefabricating multiple copies of the clonal object on a second solidsurface by providing a second solid surface having a plurality of primerpolynucleotides, hybridizing the clonal object to the primerpolynucleotides on the second solid surface, and extending the primerpolynucleotides to fabricate multiple copies of the clonal object on thesecond solid surface. The second solid surface can be located on a solidsupport that is separable from a bead or other surface used in themethods set forth herein. Generally, a first surface and second surfaceare located on separate or separable solid substrates such as differentbeads that can be separated from each other, a bead and a well that canbe separated from each other, a bead and a planar surface that can beseparated from each other, or two planar surfaces that can be separatedfrom each other. However, a first surface region and second surfaceregion can both be located on a single solid support.

In some aspects, the methods described herein include a step of primerextension or “extending” in which a polynucleotide or primer attached toa surface and/or a bead is extended using a complementary polynucleotidehybridized to the primer sequence as a template. The term “extending” orany grammatical equivalent thereof refers to making a first nucleic acidmolecule (i.e. primer or polynucleotide) longer by fabricating a copy ofa template sequence of a nucleic acid that is hybridized to the firstnucleic acid molecule. A single strand can be turned into a doublestrand by hybridizing a short sequence at one end and extending theshort sequence. The extension can be performed using a polymerase andnucleoside triphosphates or a ligase and a set of oligonucleotidecassettes of variable sequences. The extension can be carried out from auniversal primer that hybridizes to a sequence common to multipledifferent nucleic acid templates or an be carried out from a specificprimer that hybridizes to a sequence that is unique to a particulartemplate among different templates in a sample.

As used herein, the term “solid surface” is intended to mean the surfaceof a solid support or substrate and includes any material that can serveas a solid or semi-solid foundation for attachment of polynucleotides,amplicons, DNA balls, other nucleic acids and/or other polymers,including biopolymers. A solid surface of the invention can be modified,for example, to accommodate attachment of nucleic acids by a variety ofmethods well known to those skilled in the art. Exemplary types ofmaterials comprising solid surfaces include glass, modified glass,functionalized glass, inorganic glasses, microspheres, including inertand/or magnetic particles, plastics, polysaccharides, nylon,nitrocellulose, ceramics, resins, silica, silica-based materials,carbon, metals, an optical fiber or optical fiber bundles, a variety ofpolymers other than those exemplified above and multiwell microtierplates. Specific types of exemplary plastics include acrylics,polystyrene, copolymers of styrene and other materials, polypropylene,polyethylene, polybutylene, polyurethanes and Teflon™. Specific types ofexemplary silica-based materials include silicon and various forms ofmodified silicon.

Solid surfaces can also be varied in their shape depending on theapplication in a method described herein. For example, a solid surfaceuseful in the invention can be planar, or contain regions which areconcave or convex. The geometry of the concave or convex regions of thesolid surface, in some aspects of the invention, will conform to thesize and shape of the bead being attached to the surface. For example,to maximize the contact between as substantially circular bead, thecorresponding solid surface may have a diameter that is approximatelythe same as the substantially circular bead. This can be done to producea relatively large patch on the surface of the bead since the contactarea for transfer between the solid surface and bead is relativelylarge. Conversely, a relatively small patch can be produced byminimizing the contact area between a bead and a solid surface. Forexample, a bead having a convex surface can be contacted with a solidsurface that is also convex. In this case, the size of the patch will beinversely proportional to the magnitude of curvature for one or bothconvex surfaces (i.e. the surface area of the patch will decrease as thecurvature of the convex surfaces deviate from being flat). Similarly, abead having a convex surface can be contacted with a flat surface andthe size of the patch on the bead will generally be reduced as thecurvature of the bead surface increases. In another example, sphericalbeads can be contacted with a flat surface and the size of the bead canbe selected to produce a desired surface area for the patch. In thiscase, beads having a smaller diameter will generally acquire a smallerpatch than beads of a larger diameter.

In some embodiments, a solid surface that is used to form an affinitypatch on the surface of a bead can be the surface of a smaller particleor bead. For example, a single small particle can be contacted with alarger bead such that a single affinity patch is produced on the surfaceof the larger bead at the point of contact. A population of beads thateach have a single affinity patch can be created in a method wherein anexcess of beads are contacted with a scarce amount of particles suchthat, on average, each bead contacts no more than one particle. Ifdesired, the particles can have a characteristic that allows separationor enrichment of the beads that are bound to particles. For example asshown in FIG. 8, magnetic particles can be used such that particle-boundbeads can be separated from blank beads using a magnet. Separation ofthe particle bound beads can be carried out prior to, during or aftercreating the affinity patch on the surface of the bead. Anotherembodiment is shown in FIG. 9, wherein particles are in contact withbeads and further arranged on a patterned substrate.

A population of beads that are to be modified to include an affinitypatch can be contacted with particles in solution as exemplified in FIG.8. Alternatively, beads and particles can be contacted with each otheron a larger surface. For example, as shown in Figure FIG. 9, particlescan be in contact with a patterned surface and with the larger beads. Inthis example, the patterned surface can provide a desired spacingbetween particles to allow each particle to contact a single largerbead. Each of the larger beads can then be modified to include a singleaffinity patch. In a further embodiment, particles and the beads thatwill be modified to include an affinity patch can be contacted with eachother in an emulsion. An exemplary method for forming emulsion dropletseach containing a single bead and single particle is shown in FIG. 10.As shown, droplets containing individual beads can be provided in afirst emulsion stream and droplets containing individual particles canbe provided in a second fluid stream. The two fluid streams can be mixedin a converging flow device to form an emulsion of droplets containing asingle particle in contact with a single bead. If desired, the ratio ofparticles to beads can be adjusted to provide on average only a singleparticle in contact with each bead. Also, the particle can include acharacteristic that allows separation of bead bearing droplets based onthe presence or absence of a particle.

In some aspects of the above methods, the polynucleotides have auniversal primer sequence and/or a target nucleic acid molecule. Thepolynucleotides can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 300, 400 or 500 or more nucleotides. Alternatively oradditionally, the lengths are no more than 1000, 900, 800, 700, 600,500, 400, 300, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30 or 20nucleotides. In some aspects of the above methods, hybridization of thepolynucleotides on the bead to the polynucleotides on the solid surfacedefines the area for the formation of the affinity binding orhybridization patch. This area in some aspects can be less than 1000nm², or alternatively less than 500 nm², or alternatively less than 100nm². In some aspect of the above methods, the polynucleotides on thebead when hybridized to the polynucleotides of the solid surface or to aclonal object are further crosslinked together. This crosslinking canoccur through the formation of a covalent or ionic bond between thepolynucleotides and/or the clonal object.

The term “crosslinking” refers to a process of linking one molecule,such as a polymer chain to another. The bonds linking the molecules canbe covalent or ionic bonds. It can be particularly useful to covalentlycrosslink hybridized sequences so that subsequent steps that may includedenaturation of double stranded nucleic acid molecules can be used whilestill retaining the hybridized polynucleotides. A variety ofcrosslinking methods can be used so long as the crosslinking does notinhibit subsequent desired reactions with the attached nucleic acidmolecules, for example, sequencing. A particularly useful method ofcrosslinking utilizes psoralen crosslinking. Psoralen can be used toeffect crosslinking between pyrimidines on the opposite strands of ahybrid structure.

In some aspects of the methods described herein, a capture moiety, suchas a polynucleotide can have a cleavable moiety. Non-limiting examplesof cleavable moieties which are useful in the methods include proteins,nucleic acids, polynucleotides, or chemical compounds. In some aspectsof the methods, the cleavable moiety is photocleavable. A photocleavablemoiety refers to any chemical group that attaches or operably links apolynucleotide to a solid surface as described herein. Photocleavablelinkers that can be useful in the methods include, but are not limitedto, 2-nitrobenzyl moieties, alpha-substituted 2-nitrobenzyl moieties[e.g. 1-(2-nitrophenyl)ethyl moieties], 3,5-dimethoxybenzyl moieties,thiohydroxamic acid, 7-nitroindoline moieties, 9-phenylxanthyl moieties,benzoin moieties, hydroxyphenacyl moieties, and NHS-ASA moieties.Photocleavable linkers are well known to those skilled in the art (seeU.S. Pat. No. 5,739,386, and U.S. Patent Application Publication2010-0022761, both of which are herein incorporated by reference). Insome aspects, the cleavable moiety can be a sequence of nucleotidesalready present in the polynucleotide itself. For example, the cleavablemoiety can be the recognition sequence for an endonuclease, such as arestriction endonuclease, nicking endonuclease or homing endonuclease.

As used herein a “cleavable moiety” refers to a compound which isreactive to a specific catalyst, which upon reacting with the catalystreleases any bound group. Examples of cleavable moieties includecompounds that are reactive to, without limitation, proteases, enzymes,chemicals and light. In one aspect of the invention, a cleavablebase/bases could be used as the cleavable moiety, such as uracil, whichis cleavable by an exogenous base cleaving agent such as DNA glycosylase(UDG) followed by heating or chemical methods which cleave the abasicsite. Another example is a restriction enzyme motif cleavable by arestriction enzyme. Similarly, templates having 8-hydroxyguanine can becleaved by 8-hydroxyguanine DNA glycosylase (FPG protein). Otherexemplary exogenous bases and methods for their degradation that can beused are described in U.S. Patent Application Publication 2005-0181394,which is incorporated herein by reference.

Other cleavable moieties are useful for the invention including, anucleotide having a protease cleavable linker to allow selectivecleavage and removal from a solid support. As used herein, the term“protease” is intended to mean an agent that catalyzes the cleavage ofpeptide bonds in a protein or peptide. Some proteases are non-sequencespecific proteases. Generally, for the methods disclosed herein, theprotease has sequence specificity, splitting a peptide bond of a proteinbased on the presence of a particular amino acid sequence in theprotein. A protease can be characterized according to the location in aprotein where it cleaves, an endoprotease cleaving a protein betweeninternal amino acids of an amino acid chain and an exoprotease cleavinga protein to remove an amino acid from the end of an amino acid chain.In the peptide linkers of the compositions herein, an endoprotease canbe used. A protease can be characterized according to its mechanism ofaction, being identified, for example, as a serine protease, cysteine(thiol) protease, aspartic (acid) protease, metalloprotease or mixedprotease depending on the principal amino acid participating incatalysis. A protease can also be classified based on the actionpattern, examples of which include an aminopeptidase which cleaves anamino acid from the amino end of a protein, carboxypeptidase whichcleaves an amino acid from the carboxyl end of a protein, dipeptidylpeptidase which cleaves two amino acids from an end of a protein,dipeptidase which splits a dipeptide and tripeptidase which cleaves anamino acid from a tripeptide. Typically, a protease is a protein enzyme.However, non-protein agents capable of catalyzing the cleavage ofpeptide bonds in a protein, especially in a sequence specific manner arealso useful in the invention.

The term activity when used in reference to a protease is intended tomean binding of the protease to a protease substrate or hydrolysis ofthe protease substrate or both. The activity can be indicated, forexample, as binding specificity, catalytic activity or a combinationthereof. The activity of a protease can be identified qualitatively orquantitatively in accordance with the compositions and methods disclosedherein. Exemplary qualitative measures of protease activity include,without limitation, identification of a substrate cleaved in thepresence of the protease, identification of a change in substratecleavage due to presence of another agent such as an inhibitor oractivator, identification of an amino acid sequence that is recognizedby the protease, identification of the composition of a substraterecognized by the protease or identification of the composition of aproteolytic product produced by the protease. Activity can bequantitatively expressed as units per milligram of enzyme (specificactivity) or as molecules of substrate transformed per minute permolecule of enzyme (molecular activity). The conventional unit of enzymeactivity is the International Unit (IU), equal to one micromole ofsubstrate transformed per minute. A proposed coherent SystèmeInternationale (SI) unit is the katal (kat), equal to one mole ofsubstrate transformed per second.

As used herein the term, protease substrate is intended to mean amolecule that can be cleaved by a protease. A protease substrate istypically a protein, protein moiety or peptide having an amino acidsequence that is recognized by a protease. A protease can recognize theamino acid sequence of a protease substrate due to the specific sequenceof side chains or due to properties generic to proteins. A proteasesubstrate can also be a protein mimetic or non-protein molecule that iscapable of being cleaved or otherwise covalently modified by a protease.

Exemplary proteases, corresponding peptide substrates and theircommercial sources are shown in Table 1.

TABLE 1 Proteases and their cleavage preferences. Peptide (cleavage siteindicated Protease with dash) Company Thrombin LVPR-GSAmersham, Novagen, Sigma, Roche Factor Xa IEGR-X Amersham, NEB, RocheEnterokinase DDDDK-X NEB, Novagen, Roche TEV protease ENLYFQ-GInvitrogen PreScission LEVLFQ-GP Amersham HRV 3C Protease LEVLFQ-GPNovagen Trypsin R-X, K-X Endoproteinase X-D Asp-N Chymotrypsin Y-X, F-X,W-X Endoproteinase E-X Glu-C Endoproteinase R-X Arg-C Endoproteinase K-XLys-C

Protease cleavable linkers used in the invention are generally peptides.Peptide synthesis can be carried out using standard solid phase orsolution phase chemistry, as desired. Methods for peptide synthesis arewell known to those skilled in the art (Fodor et. al., Science 251:767(1991); Gallop et al., J. Med. Chem. 37:1233-1251 (1994); Gordon et al.,J. Med. Chem. 37:1385-1401 (1994)). It is understood that a peptidelinker can be synthesized and then added to the NTP as a peptide or canbe synthesized by sequentially adding amino acids and then a dye.

Embodiments of the invention provide a method of amplifying a targetnucleic acid molecule by placing the compositions or populations ofbeads having affinity bound or hybridized clonal objects describedherein onto a solid surface having microwells, wherein only one bead canspatially fit into one microwell and amplifying the target nucleic acidmolecules in the microwells, thereby forming amplicons. In some aspectsof the invention, the method further includes determining the nucleicacid sequence of the target nucleic acid molecule. In one aspect,determining the nucleic acid sequence of the target nucleic acidmolecule includes sequencing the amplified target nucleic acidmolecules, thereby determining the nucleic acid sequence of the targetnucleic acid molecule.

In some aspects, determining the nucleic acid sequence of the targetnucleotide molecule further includes quantifying the target nucleic acidmolecule or amplicons. Methods for quantifying a target nucleic acidmolecule or amplicon are well known to one of skilled in the art. Forexample, during amplification of the target nucleic acid, quantitativetechniques such as real-time polymerase chain reaction (RT-PCR) can beused to quantify the copy number of target nucleic acid moleculespresent in the clonal object as discussed in Logan et al. Real-Time PCR:Current Technology and Applications, Caister Academic Press. (2009).Briefly, RT-PCR follows the general principle of polymerase chainreaction, however inclusion of detection molecules, such as non-specificfluorescent dyes that intercalate with any double-stranded DNA, orsequence-specific DNA probes consisting of oligonucleotides that arelabeled with a fluorescent reporter, which permits detection only afterhybridization of the probe with its complementary DNA target, allows forthe detection of nucleic acid formed during amplification. The rate ofdetectable molecules is proportional to the copy number of targetnucleic acid molecules present in the clonal object. Furthermore,quantifying the target nucleic acid molecule or amplicons can be donefollowing amplification using standard gel electrophoresis and/orSouthern blot techniques, which are well practiced in the art.

In some aspects, the method includes hybridizing a probe nucleic acid tothe amplicon, thereby identifying the target nucleic acid molecule.Methods of hybridizing a probe nucleic acid to identify the targetnucleic acid molecule are well know to one skilled in the art andexamples of such methods are described here. For example, anoligonucleotide ligation assay (OLA), as described below, is a methodthat utilizes hybridizing a probe to identify the target nucleic acidmolecule.

In some aspects of the method, the solid surface is a microarray, whichcan have microwells having a diameter sufficient to allow only one beadhaving the clonal object into the well. It is understood that the sizeof the microwell will be dependent upon the size of the bead and/or thesize of the clonal object. In some aspects of the invention, thediameter of the microwells are less than 200 μm, or alternatively lessthan 100 μm, or alternatively less than 50 μm, or alternatively lessthan 40 μm, or alternatively less than 30 μm, or alternatively less than20 μm, or alternatively less than 10 μm, or alternatively less than 5μm. It is also understood that the size of the microwells on themicroarray can be of various sizes and will ultimately depend on thesystems and/or apparatus used to analyze later reactions.

In some aspects, the method includes amplifying the clonal object or insome aspects the target nucleic acid molecules present in the clonalobject. Methods of amplifying nucleic acid sequences are well know toone of skill in the art. Particularly useful methods for amplifying thetarget nucleic acids includes, but is not limited to, solid-phase clonalamplification. Solid-phase clonal amplification can be done using anumber of PCR techniques known in the art such as bridge amplificationusing two or more primer polynucleotides immobilized on a bead or solidsurface. Useful bridge amplification methods include those where one orboth of the primers used for amplification are attached to a solid phaseas described, for example, in US 2008/0286795; US 2007/0128624 and US2008/0009420, each of which is incorporated herein by reference. Anotheruseful method for solid-phase clonal amplification is multipledisplacement amplification.

In some aspects, the methods for amplifying a target nucleic acidmolecule further includes cleaving between the tandem repeats of thesingle tandemly repeated target nucleic acid molecule, i.e. DNA ball orcircular library element, prior to amplifying the target nucleic acidmolecules. This cleaving step generates a population of target nucleicacid molecules, which can be more readily accessible to lateramplification reactions, such as PCR or bridge amplification methods. Insome aspects of the method, the cleavage step includes hybridizing anoligonucleotide to the clonal object and cleaving the clonal object byan enzyme, such as, but not limited to, a homing endonuclease, arestriction endonuclease or a nicking endonuclease. These endonucleasesare well known to those skilled in the art and are available fromseveral sources (New England Biolabs—Ipswich, Mass.; PromegaCorporation—Madison, Wis. and Life Technologies—Carlsbad, Calif.).

The expression “amplification” or “amplifying” includes methods such asPCR, ligation amplification (or ligase chain reaction, LCR), multipledisplacement amplification (MDA) and other amplification methods. Thesemethods are known and widely practiced in the art. See, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202 and Innis et al., “PCR protocols: a guideto method and applications” Academic Press, Incorporated (1990) (forPCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR). In general, thePCR procedure describes a method of gene amplification which iscomprised of (i) sequence-specific hybridization of primers to specificgenes within a DNA sample (or library), (ii) subsequent amplificationinvolving multiple rounds of annealing, elongation, and denaturationusing a DNA polymerase, and (iii) screening the PCR products for a bandof the correct size. The primers used are oligonucleotides of sufficientlength and appropriate sequence to provide initiation of polymerization,i.e. each primer is specifically designed to be complementary to eachstrand of the genomic locus to be amplified.

Amplification methods set forth herein can be carried out on the surfaceof a bead or on the surface of an array substrate following transfer ofthe bead to the array. For example, a bead having a clonal object can betransferred to a well in an array of wells and the clonal object can beamplified in the well. The amplification method can include those whereone or both of the primers used for amplification are attached to thebead or to the surface of the array, for example, the inner surface of awell. In particular embodiments, MDA primers can be attached to thesurface of a bead, well or other surface such that the MDA primers comeinto contact with a clonal object and the clonal object is amplified.The resulting amplicons can be concatameric copies of the clonal objectthat are attached to the bead, well or other surface. Amplification neednot be carried out using solid phase primer(s) and can instead becarried out in solution such that different target sequences areseparated from each other by isolation in a well, emulsion droplet orother reaction vessel. In some embodiments amplification can includeboth the use of solid phase amplification primer(s) as well as isolationof target nucleic acids in a well, emulsion droplet or other reactionvessel.

Reagents and hardware for conducting amplification reaction arecommercially available. Primers useful to amplify sequences from aparticular gene region are preferably complementary to, and hybridizespecifically to sequences in the target region or in its flankingregions and can be prepared using the polynucleotide sequences providedherein. Nucleic acid sequences generated by amplification may besequenced directly. Alternatively the amplified sequence(s) may becloned prior to sequence analysis. Methods for the direct cloning andsequence analysis of enzymatically amplified genomic segments are knownin the art.

One method to eliminate primer-dimer interactions, which are oftenassociated with traditional PCR, is to perform solid-phase PCR usingprimer pairs physically separated on beads as a multiplex bridge PCRreaction (Adams et al., U.S. Pat. No. 5,641,658). Each primer set can beindividually co-immobilized and then later all the beads are mixedtogether to form one grand master mix. This master bead mix can beinoculated into the PCR mix along with all the other PCR components andtarget DNA. Key parameters in the solid-phase amplification reaction canbe varied including, but not limited to, linker length between theprimer and beads. After amplification, the library elements can becleaved from the beads and processed as a standard library forgeneration of clonal arrays.

In some aspects, the method for determining the nucleic acid sequence ofa target nucleic acid molecule includes sequencing, which is well knownto one skilled in the art. In some aspects, sequencing by synthesis,sequencing by ligation or sequencing by hybridization is used fordeterring the nucleic acid sequence of a target nucleic acid molecule.Nucleic acid sequencing has become an important technology withwidespread applications, including mutation detection, whole genomesequencing, exon sequencing, mRNA or cDNA sequencing, alternatetranscript profiling, rare variant detection, and clone counting,including digital gene expression (transcript counting) and rare variantdetection. As disclosed herein, various amplification methods can beemployed to generate larger quantities, particularly of limited nucleicacid samples, prior to sequencing. For example, the amplificationmethods can produce a targeted library of amplicons. The ampliconswhether or not they are targeted amplicons can be in the form of DNAballs.

Two useful approaches for high throughput or rapid sequencing aresequencing by synthesis (SBS) and sequencing by ligation. Target nucleicacid of interest can be amplified, for example, using ePCR, as used by454 Lifesciences (Branford, Conn.) and Roche Diagnostics (Basel,Switzerland). Nucleic acid such as genomic DNA or others of interest canbe fragmented, dispersed in water/oil emulsions and diluted such that asingle nucleic acid fragment is separated from others in an emulsiondroplet. A bead, for example, containing multiple copies of a primer,can be used and amplification carried out such that each emulsiondroplet serves as a reaction vessel for amplifying multiple copies of asingle nucleic acid fragment. Other methods can be used, such asbridging PCR (Illumina, Inc.; San Diego Calif.), or polony amplification(Agencourt/Applied Biosystems).

For sequencing by ligation, labeled nucleic acid fragments arehybridized and identified to determine the sequence of a target nucleicacid molecule. For sequencing by synthesis (SBS), labeled nucleotidescan be used to determine the sequence of a target nucleic acid molecule.A target nucleic acid molecule can be hybridized with a primer andincubated in the presence of a polymerase and a labeled nucleotidecontaining a blocking group. The primer is extended such that thenucleotide is incorporated. The presence of the blocking group permitsonly one round of incorporation, that is, the incorporation of a singlenucleotide. The presence of the label permits identification of theincorporated nucleotide. Either single bases can be added or,alternatively, all four bases can be added simultaneously, particularlywhen each base is associated with a distinguishable label. Afteridentifying the incorporated nucleotide by its corresponding label, boththe label and the blocking group can be removed, thereby allowing asubsequent round of incorporation and identification. Thus, it isdesirable to have conveniently cleavable linkers linking the label tothe base, such as those disclosed herein, in particular peptide linkers.Additionally, it is advantageous to use a removable blocking group sothat multiple rounds of identification can be performed, therebypermitting identification of at least a portion of the target nucleicacid sequence. The compositions and methods disclosed herein areparticularly useful for such an SBS approach. In addition, thecompositions and methods can be particularly useful for sequencing froman array, where multiple sequences can be “read” simultaneously frommultiple positions on the array since each nucleotide at each positioncan be identified based on its identifiable label. Exemplary methods aredescribed in US 2009/0088327; US 2010/0028885; and US 2009/0325172, eachof which is incorporated herein by reference.

The oligonucleotides, nucleosides and nucleotides described herein canbe particularly useful for nucleotide sequence characterization orsequence analysis. Reversible labeling, reversible termination or acombination thereof can allow accurate sequencing analysis to beefficiently performed. Methods for manual or automated sequencing arewell known in the art and include, but are not limited to, Sangersequencing, Pyrosequencing, sequencing by hybridization, sequencing byligation and the like. Sequencing methods can be preformed manually orusing automated methods. Furthermore, the amplification methods setforth herein can be used to prepare nucleic acids for sequencing usingcommercially available methods such as automated Sanger sequencing(available from Applied Biosystems, Foster City, Calif.) orPyrosequencing (available from 454 Lifesciences, Branford, Conn. andRoche Diagnostics, Basel, Switzerland); for sequencing by synthesismethods commercially available from Illumina, Inc. (San Diego, Calif.)or Helicos (Cambridge, Mass.) or sequencing by ligation methods beingdeveloped by Applied Biosystems in its Agencourt platform (see alsoRonaghi et al., Science 281:363 (1998); Dressman et al., Proc. Natl.Acad. Sci. USA 100:8817-8822 (2003); Mitra et al., Proc. Natl. Acad.Sci. USA 100:55926-5931 (2003)).

A population of nucleic acids, such as DNA balls or other amplicons setforth herein, can be sequenced using methods in which a primer ishybridized to each nucleic acid such that the nucleic acids formtemplates and modification of the primer occurs in a template directedfashion. The modification can be detected to determine the sequence ofthe template. For example, the primers can be modified by extensionusing a polymerase and extension of the primers can be monitored underconditions that allow the identity and location of particularnucleotides to be determined. For example, extension can be monitoredand sequence of the template nucleic acids determined usingPyrosequencing which is described in U.S. Patent ApplicationPublications 2005/0130173 and 2006/0134633 and U.S. Pat. Nos. 4,971,903;6,258,568 and 6,210,891, each of which is incorporated herein byreference, and is also commercially available. Extension can also bemonitored according to addition of labeled nucleotide analogs by apolymerase, using methods described, for example, in U.S. Pat. No.4,863,849; U.S. Pat. No. 5,302,509; U.S. Pat. No. 5,763,594; U.S. Pat.No. 5,798,210; U.S. Pat. No. 6,001,566; U.S. Pat. No. 6,664,079; U.S.2005/0037398; and U.S. Pat. No. 7,057,026, each of which is incorporatedherein by reference. Polymerases useful in sequencing methods aretypically polymerase enzymes derived from natural sources. It will beunderstood that polymerases can be modified to alter their specificityfor modified nucleotides as described, for example, in WO 01/23411; U.S.Pat. No. 5,939,292; and WO 05/024010, each of which is incorporatedherein by reference. Furthermore, polymerases need not be derived frombiological systems. Polymerases that are useful in the invention includeany agent capable of catalyzing extension of a nucleic acid primer in amanner directed by the sequence of a template to which the primer ishybridized. Typically polymerases will be protein enzymes isolated frombiological systems.

A further modification of primers that can be used to determine thesequence of templates to which they are hybridized is ligation. Suchmethods are referred to as sequencing by ligation and are described, forexample, in Shendure et al. Science 309:1728-1732 (2005); U.S. Pat. No.5,599,675; and U.S. Pat. No. 5,750,341, each of which is incorporatedherein by reference. It will be understood that primers need not bemodified in order to determine the sequence of the template to whichthey are attached. For example, sequences of template nucleic acids canbe determined using methods of sequencing by hybridization such as thosedescribed in U.S. Pat. No. 6,090,549; U.S. Pat. No. 6,401,267 and U.S.Pat. No. 6,620,584. It is understood that many of the uses ofcompositions of the present invention can be applied to both sequencingby synthesis (SBS) or single base extension (SBE), since both utilizeextension reactions.

A DNA ball or other amplicons produced using methods set forth hereincan be used in an extension assay. Extension assays are useful fordetection of alleles, mutations or other nucleic acid features in anamplicon of interest. Extension assays are generally carried out bymodifying the 3′ end of a first nucleic acid when hybridized to a secondnucleic acid such as a DNA ball or other amplicon. The amplicon can actas a template directing the type of modification, for example, by basepairing interactions that occur during polymerase-based extension of thefirst nucleic acid to incorporate one or more nucleotide. Polymeraseextension assays are particularly useful, for example, due to therelative high-fidelity of polymerases and their relative ease ofimplementation. Extension assays can be carried out to modify nucleicacid probes that have free 3′ ends, for example, when bound to asubstrate such as an array. Exemplary approaches that can be usedinclude, for example, allele-specific primer extension (ASPE), singlebase extension (SBE), degenerate probe ligation such as that used in theSOLiD system sold by Life Technologies (Carlsbad, Calif.) orPyrosequencing as described, for example, in U.S. 2005/0181394, which isincorporated herein by reference. A nucleic acid, nucleotide ornucleoside having a reversible blocking group on a 2′, 3′ or 4′hydroxyl, a peptide linked label or a combination thereof can be used insuch methods. For example the nucleic acid, nucleotide or nucleoside canbe included in the first nucleic acid or the second nucleic acid.Additionally or alternatively, the nucleic acid, nucleotide ornucleoside can be used to modify the free 3′ ends in the extensionreactions.

In particular embodiments, single base extension (SBE) can be used fordetection of an allele, mutations or other nucleic acid features. Thecompositions of the present invention are useful in an SBE method, inparticular, a nucleoside or nucleotide containing a peptide linker,allowing cleavage and removal of a label, and/or terminator blockinggroup, either removable or non-removable. Briefly, SBE utilizes anextension probe that hybridizes to a target genome fragment at alocation that is proximal or adjacent to a detection position, thedetection position being indicative of a particular locus. A polymerasecan be used to extend the 3′ end of the probe with a nucleotide analoglabeled with a detection label such as those described previouslyherein. Based on the fidelity of the enzyme, a nucleotide is onlyincorporated into the extension probe if it is complementary to thedetection position in the target nucleic acid. If desired, thenucleotide can be derivatized such that no further extensions can occurusing a blocking group, including reversible blocking groups, and thusonly a single nucleotide is added. The presence of the labelednucleotide in the extended probe can be detected for example, at aparticular location in an array and the added nucleotide identified todetermine the identity of the locus or allele. SBE can be carried outunder known conditions such as those described in U.S. patentapplication Ser. No. 09/425,633. A labeled nucleotide can be detectedusing methods known to one of skill in the art, such as those describedin Syvanen et al., Genomics 8:684-692 (1990); Syvanen et al., HumanMutation 3:172-179 (1994); U.S. Pat. Nos. 5,846,710 and 5,888,819; andPastinen et al., Genomics Res. 7(6):606-614 (1997).

ASPE is an extension assay that utilizes extension probes that differ innucleotide composition at their 3′ end. An ASPE method can be performedusing a nucleoside or nucleotide containing a cleavable linker, so thata label can be removed after a probe is detected. This allows furtheruse of the probes or verification that the signal detected was due tothe label that has now been removed. Briefly, ASPE can be carried out byhybridizing a sample nucleic acid, or amplicons derived therefrom, to anextension probe having a 3′ sequence portion that is complementary to adetection position and a 5′ portion that is complementary to a sequencethat is adjacent to the detection position. Template directedmodification of the 3′ portion of the probe, for example, by addition ofa labeled nucleotide by a polymerase yields a labeled extension product,but only if the template includes the target sequence. The presence ofsuch a labeled primer-extension product can then be detected, forexample, based on its location in an array to indicate the presence of aparticular allele.

In particular embodiments, ASPE can be carried out with multipleextension probes that have similar 5′ ends such that they annealadjacent to the same detection position in a target nucleic acid butdifferent 3′ ends, such that only probes having a 3′ end thatcomplements the detection position are modified by a polymerase. A probehaving a 3′ terminal base that is complementary to a particulardetection position is referred to as a perfect match (PM) probe for theposition, whereas probes that have a 3′ terminal mismatch base and arenot capable of being extended in an ASPE reaction are mismatch (MM)probes for the position. The presence of the labeled nucleotide in thePM probe can be detected and the 3′ sequence of the probe determined toidentify a particular allele at the detection position.

A sequence or allele present in an amplicon, such as a DNA ball, can bedetected using a ligation assay such as oligonucleotide ligation assay(OLA). Detection with OLA involves the template-dependent ligation oftwo smaller probes into a single long probe, using a target sequence inan amplicon as the template. In a particular embodiment, asingle-stranded target sequence includes a first target domain and asecond target domain, which are adjacent and contiguous. A first OLAprobe and a second OLA probe can be hybridized to complementarysequences of the respective target domains. The two OLA probes are thencovalently attached to each other to form a modified probe. Inembodiments where the probes hybridize directly adjacent to each other,covalent linkage can occur via a ligase. One or both probes can includea nucleoside having a label such as a peptide linked label. Accordingly,the presence of the ligated product can be determined by detecting thelabel. In particular embodiments, the ligation probes can includepriming sites configured to allow amplification of the ligated probeproduct using primers that hybridize to the priming sites, for example,in a PCR reaction.

Alternatively, the ligation probes can be used in an extension-ligationassay wherein hybridized probes are non-contiguous and one or morenucleotides are added along with one or more agents that join the probesvia the added nucleotides. Furthermore, a ligation assay orextension-ligation assay can be carried out with a single padlock probeinstead of two separate ligation probes. The ends of the padlock probeare designed to complement adjacent or proximal sequence regions in anamplicon or other template such that ligation or extension followed byligation results in a circularized padlock probe. The probe can beamplified by rolling circle amplification. Exemplary conditions forligation assays or extension-ligation assays using separate probes orligation probes are described, for example, in U.S. Pat. No. 6,355,431B1 and U.S. 2003/0211489, each of which is incorporated herein byreference.

A ligation probe such as a padlock probe used in the invention canfurther include other features such as an adaptor sequence, restrictionsite for cleaving concatemers, a label sequence or a priming site forpriming an amplification reaction as described, for example, in U.S.Pat. No. 6,355,431 B1.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Loading Microbeads with Single Stranded Targets for DNASequencing

Certain types of DNA sequencing techniques, such as Pyrosequencing, whenutilized in a high throughput multiwell system require that sufficientsingle stranded target DNA is present in each microwell in order toproduce a robust signal and in the case of Pyrosequencing for everycycle of dNTP addition. Furthermore, each microwell must typicallycontain only a single DNA target sequence with several copies of thetarget sequence in order to have a clear signal above any detectablebackground. The following method described herein achieves both of theserequirements for a system designed to perform both PCR and sequencing onthe same platform.

Briefly, the following method is based on rolling circle amplification(RCA) to produce DNA balls from discreet DNA sequences. DNA balls arethen bound to primer loaded microbeads that contain a binding spot of asufficient size that excludes the binding of more than one DNA ball permicrobead. After the DNA balls have been attached to the microbeads, themicrobeads are placed into the microwells of a PCR/sequencing platform.The multiple copies of the tandemly repeated target sequences are thencut to produce numerous single stranded DNA target sequences. Theseidentical target sequences have primer sequences (P1′), which arecomplementary to the primers (P1) attached to the microbead, and theprimer sequence (P2). P1′ sequences are annealed to P1 primers on thebead and PCR is performed with P2 primers in solution. Afteramplification by PCR, all DNA fragments, which are not covalentlyattached to the beads are removed. After this step, the beads are readyfor sequencing.

Production of Microbeads with Affinity Binding Patch

Polynucleotide primers having a P1 sequence are attached to microbeadsusing methods described in U.S. Pat. No. 7,259,258 (incorporated hereinby reference) thereby generating a pool of charged microbeads (FIG. 1).The preferred size of the microbeads for generating the desired affinitybinding patch is 10-15 μm for attaching an RCA product having 100-1000copies of the target sequence (˜200-500 nm patch diameter). However, thesize of the beads can be varied depending on the length of the primersattached to the bead and the size of the DNA ball to be attached. Aplate charged with biotinylated primers having a complementary P1′sequence are generated using microfabrication lithography (FIG. 1). Thebiotinylated primers are attached to the plate through a linker whichhas a cleavable moiety, such as a photocleavable linking group. Next thecharged microbeads are hybridized to the surface of the plate.

Once the charged beads have been immobilized to the surface of theplate, the hybridized primers can be optionally crosslinked to form amore stable bond between the two primers. Crosslinking of the primers isdone through introduction of psoralen. Psoralen can be introduced to aprimer by incorporating a psoralen labeled T phosphoramidite duringstandard phosphodiester oligonucleotide synthesis chemistry. Next theimmobilized beads are released from the plate by cleaving the linkerwith light of an appropriate wavelength to cleave the photochemicallinker. The unbound P1′ primers are separated from the releasedmicrobeads, generating a P1 charged microbead with a biotin charged spotlocated at one region of the bead (FIG. 2).

The beads generated above are then challenged with streptavidintetramers. The streptavidin tetramers are capable of binding more thanone biotin and thus after the spot is charged with streptavidin, thebeads are now capable of binding to another biotin molecule (FIG. 3).The beads now contain an affinity binding patch of streptavidintetramers capable of binding a biotinylated DNA ball.

Generation of DNA Balls

Target DNA molecules are generated by adapter ligation approaches suchas those described in US 2007/0128624 and US 2008/0009420 (each of whichis incorporated herein by reference) to have P1 and P2′ primer sequenceattached to the 5′ and 3′ ends of the DNA molecule, respectively. Thetarget DNA molecules are then denatured by heat and/or 0.1 N NaOH togenerate single stranded molecules. The single stranded target moleculesare hybridized to splint primer polynucleotides having the complementaryprimer sequences P1′ and P2 (FIG. 4). The splint primer polynucleotidescan also contain one or more biotinylated nucleotide for later bindingto the microbeads. Additionally, the P1′ and P2 sequences can beconstructed to contain a restriction enzyme or top nicking endonucleaserecognition sequence for later cleavage (FIG. 5). Using the splint togenerate a double stranded DNA molecule, the complementary ends of thesingle stranded target molecules are ligated to each other by T4 DNAligase forming a single stranded DNA template (FIG. 4).

After ligation, the splint is used as the primer in rolling circleamplification (RCA) to produce a DNA ball. RCA is conducted by extendingthe splint primer using phi29 polymerase and nucleotides. A lowpercentage of biotinylated dNTPs can be included in this amplificationreaction in order to label 10% or fewer of the tandem sequences in theDNA ball. Incorporation of the these additional biotinylated dNTPs willfacilitate efficient binding to the affinity binding patch of themicrobead by increasing the likelihood that a biotin molecule is exposedto the outer surface of the DNA ball.

Amplification and Sequencing of Target DNA

Microbeads containing an affinity binding spot as described above areattached to the DNA balls by binding of the biotin on the splint primer(which was incorporated into the DNA ball during RCA) to thestreptavidin molecules at the patch. More specifically, the streptavidinacts as an intermediary to bind the DNA ball to the beads because thestreptavidin molecules that are bound to the patches (as shown in FIG.3) are tetrameric and can therefore bind to the biotin on the P1 chargedbead and to the biotin on the DNA ball.

After the DNA balls have been attached to the microbeads (one permicrobead), the microbeads are placed into the microwells of thePCR/sequencing platform by random deposition as described for example inU.S. Pat. No. 7,622,294, which is incorporated herein by reference. Thesize and shape of the microwells is such that only one microbead can fitinto a microwell. The microwells are also separated from each other sothat PCR and sequencing can be done in each well.

Prior to amplification of the target DNA molecule, the tandem sequencesare cleaved from each other. This is done by hybridizing anoligonucleotide that is complementary to the region of the P1′/P2sequence, which contain the restriction endonuclease (RE) or top nickingendonuclease recognition sequence (FIG. 6). The double stranded regionsare then cleaved by incubation with a site specific nuclease therebyreleasing the tandem sequences at the boundary between the P1′ and P2sequences. Many restriction endonucleases are ideally suited for thisstep because they only cut double stranded structures.

Amplification of the released single stranded tandem sequences occurs bysolid-phase assisted PCR. The resulting single stranded sequences arehybridized to the P1 primers on the microbead to provide multipletargets for in-well PCR. Following amplification by PCR, the unattachedfragments are washed away and the single stranded fragments on the beadsare ready for DNA sequencing. One advantage of the above method is thatthe amplification of the target sequence only requires P2 primers andother standard PCR ingredients for successful in-well PCR.

Sequencing of the target DNA molecule can be done using a variety ofknown methods, including Pyrosequencing. Pyrosequencing of the targetDNA molecule is conducted by using methods described in US 2009/0286299.

Example II Loading Microbeads with DNA Balls Using PatternedPolynucleotide Patches on a Chip

The following method describes utilizing the surface of a chip thatcontains patterned polynucleotides for loading microbeads with DNA ballsat a desired ratio. Using the described method, single 100-1000 nmactive patches are created on a larger microsphere (5-50 μm) such thatthe active patch can capture a single DNA ball. In this manner, when themicrospheres are incubated with DNA balls, the result is that everymicrosphere has one and only one DNA ball attached to it to allow forlater clonal amplification. The seeding of a DNA ball rather than asingle library molecule on a microsphere greatly improves the signal tonoise in the amplification reaction.

A chip containing patterned polynucleotides patches is generated bymicrofabrication lithography. The polynucleotides on the chip contain acleavable moiety such as uracil to allow for later release from thesurface of the chip (FIG. 7). DNA balls containing the desired targetnucleic acid molecules are seeded to the surface of the chip byannealing (FIG. 7). Alternatively, DNA balls are generated using themethod described in Example I having biotin molecules at the ends orwithin the ball itself. These DNA balls can be seeded to chips patternedwith streptavidin by microfabrication lithography. The patches ofpolynucleotides or streptavidin are 200-500 nm in size, which stericallyallows for attaching a single DNA ball having 100-1000 copies of thetarget sequence. Once the DNA balls are seeded to the chip, microspherescontaining attached polynucleotides are attached to the DNA balls byhybridization to a sequence present in an adapter region. Theimmobilized balls are then released from the chip by cutting thecleavable moiety attaching the polynucleotides to the chip (FIG. 7).Cutting of the cleavable moiety is performed by Uracil-Specific ExcisionReagent (USER available from New England Biolabs, USA).

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1-77. (canceled)
 78. A method of fabricating a bead having one clonalobject comprising: (a) providing a bead comprising a plurality of firstpolynucleotides; (b) providing a solid surface comprising a plurality ofsecond polynucleotides patterned into patches on said surface, whereinsaid second polynucleotides each comprise a cleavable moiety, whereinone clonal object is hybridized to one polynucleotide patch on saidsurface, and wherein said one clonal object comprises a single tandemlyrepeated target nucleic acid molecule; (c) hybridizing said firstpolynucleotides to said clonal object, thereby forming an immobilizedbead on the solid surface, and (d) cleaving said second polynucleotidesat said cleavable moiety so as to retain said clonal object, therebyfabricating a bead having one clonal object.
 79. The method of claim 78,further comprising fabricating multiple copies of said clonal object ona second solid surface comprising: (e) providing said second solidsurface comprising a plurality of primer polynucleotides; (f)hybridizing said clonal object to said primer polynucleotides on saidsecond solid surface, and (g) extending said primer polynucleotides tofabricate multiple copies of said clonal object on said second solidsurface.
 80. The method of claim 78, wherein said first polynucleotidescomprise a universal primer sequence.
 81. The method of claim 78,wherein said first polynucleotides comprise a target nucleic acidmolecule.
 82. The method of claim 78, wherein said secondpolynucleotides comprise a universal primer.
 83. The method of claim 78,wherein said solid surface is planar.
 84. The method of claim 78,wherein said solid surface comprises regions which are concave orconvex.
 85. The method of claim 78, wherein said first or secondpolynucleotides have a length selected from the group consisting of atleast 10, 20, 30, 40 and 50 nucleotides.
 86. The method of claim 78,wherein said first or second polynucleotides have a length of at least10 nucleotides and no more than 500 nucleotides.
 87. The method of claim78, wherein said immobilization step is followed by crosslinking saidfirst polynucleotides to said clonal object.
 88. The method of claim 87,wherein said crosslinking occurs through a covalent or ionic bond. 89.The method of claim 78, wherein said cleavable moiety is cleavable by aprotease, an enzyme or a chemical.
 90. The method of claim 78, whereinsaid cleavable moiety is photocleavable.
 91. The method of claim 78,wherein said clonal object is a DNA ball.
 92. The method of claim 91,wherein said DNA ball is produced by rolling circle amplification or DNAligation.
 93. The method of claim 78, wherein said clonal objectcomprises multiple copies of said single tandemly repeated targetnucleic acid molecule selected from the group consisting of at least100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 and10,000 copies.
 94. The method of claim 78, wherein said clonal objecthas a diameter selected from the group consisting of 0.1 μm, 0.2 μm, 0.5μm, 1 μm, 2 μm, 3 μm, 4 μm and 5 μm.
 95. A method of fabricating ahybridization patch on a bead comprising: (a) providing a beadcomprising a plurality of first polynucleotides attached to the surfaceof said bead, wherein the first polynucleotides each comprise a firstcapture sequence; (b) providing a solid surface comprising a pluralityof second polynucleotides attached to said solid surface, wherein saidsecond polynucleotides each comprise a first capture-complement sequenceand a second capture-complement sequence; (c) hybridizing said firstcapture sequences of said first polynucleotides to said firstcapture-complement sequence of said second polynucleotides, therebyforming an immobilized bead on the solid surface, and (d) extending saidfirst polynucleotides of said immobilized bead using said secondcapture-complement sequence as a template, thereby fabricating ahybridization patch of extended first polynucleotides on said bead, saidextended first polynucleotides comprising a second capture sequence.